The past several decades have witnessed exponential progress in the health sciences and biotechnology fields. From the mapping of the human genome to the development of proteomics, today's scientific community has gained an understanding of the processes of living organisms unimaginable to earlier generations. At the core of this progress has been the development of new instruments and new techniques that have allowed scientists to correctly analyze and identify the molecules that are central to their scientific inquiry. To continue to push their progress forward, researchers in these fields are in constant in need of new instruments and/or analytical techniques that offer more precise analysis of these molecules.
One difficulty confronting current researchers is created by the fact that molecules that have structural differences can appear similar or identical in a given analysis. For example, when analyzed in a mass spectrometer, different isoforms of the same protein present the same m/z peaks, and can thus appear identical despite their structural differences.
This problem is illustrated in top-down proteomics, which may involve direct analysis of intact proteins, access to complete protein sequences, and localization and characterization of post translational modifications (PTMs) of the intact proteins. PTMs have well-documented roles in signal transduction, regulation of cellular processes, clinical biomarkers, and therapeutic targets. The identification of proteins with similar or identical mass to charge ratios thus provides an excellent example of the more general problems facing scientists seeking to provide devices and techniques that allow high resolution characterization of molecules.
One exemplary system of such analysis is a so-called Orbitrap mass spectrometer (MS). Due to high mass resolution and mass accuracy at higher molecular masses, an Orbitrap MS is an attractive detection system for biochemical analysis of intact proteins. The high resolution high mass accuracy of the Orbitrap MS provides high sensitivity detection and reliable identification of protein isoforms. However, as is the case with many MS systems, an Orbitrap MS system will not necessarily distinguish between two isoforms of the same protein. It is the different isoforms of the same protein that yield biologically significant functions, and are implicated in a variety of diseases, including Alzheimer's and cancer.
Another exemplary device used in sample analysis is a Drift Tube Ion Mobility Spectrometry (IMS). An IMS is an orthogonal to MS gas phase separation approach, which differentiate analytes by their shapes and is capable of distinguishing different protein isoforms. However, while an IMS can provide information distinguishing different protein isoforms, it does not provide high resolution and high mass accuracy of the Orbitrap MS.
The coupling of an Orbitrap system with in IMS system would appear to offer ideal separation combined with high resolution detection. However, prior to the present invention, no successful coupling of these devices has occurred, which illustrates a more general problem.
Coupling and efficient operation of a relatively fast separation system, such as an IMS, with a relatively slow MS detection system, such as an Orbitrap system, is problematic because of the different acquisition timescales. Specifically, an IMS separation proceeds relatively rapidly when compared with the analysis in the Obritrap system. Thus, while IMS instrument enables separation of different protein isoforms by their tertiary structures, a feature unavailable with current MS instrumentation, the direct injection of these analytes from the IMS to, for example, and Orbitrap MS would lose this separation data because separated ions would simply be recombined in the Orbitrap MS, due to its relatively slow acquisition time.
In contrast, in systems where a relatively slow separation is coupled with a relatively fast MS analysis, the problem does not exist because the acquisition period of the detection system is shorter than the temporal profile of a front-end separation peak, so that multiple detector measurements can be performed for any given separation peak. The fundamental issue in coupling any fast separation technique (e.g., IMS) to slower detection system (e.g., the Orbitrap or FTICR MS) is the inability to acquire a separation spectrum, as the separation completes before acquisition of a single mass spectrum.
Thus, there is a need for the effective integration and coupling of relatively fast separation techniques with relatively slower MS analysis systems. There is a further need for the ability to analyze samples wherein both the mass spectrometry and the mobility data are preserved, such that the final output is an accurate mass spectrum that retains the high resolution data from every digitized point where each ion species is identified not only by the mass to charge ratio, but also by the separation time of the various species in the sample. The present invention meets those needs.